The mechanism of action of yeast pyruvate decarboxylase will be studied by a variety of techniques. First, novel approaches to enzyme purification will be attempted employing affinity chromatography. The binding constants of the cofactors to the apoenzyme will be next determined employing fluorescence titrations. C-13 kinetic isotope effects will be determined for the decarboxylation of pyruvate at the C1 and C2 atoms., the latter by converting the resulting acetaldehyde to CO2. Model studies on thiamin catalyzed pyruvate decarboxylation will also be performed to probe the effect of coenzyme conformational aspects on the elementary reaction steps believed to occur in the enzymatic process.